RhoB Protects Human Keratinocytes from UVB-induced Apoptosis through Epidermal Growth Factor Receptor Signaling

Abstract

Exposure of the skin to UVB light results in the formation of DNA photolesions that can give rise to cell death, mutations, and the onset of carcinogenic events. Specific proteins are activated by UVB and then trigger signal transduction pathways that lead to cellular responses. An alteration of these signaling molecules is thought to be a fundamental event in tumor promotion by UVB irradiation. RhoB, encoding a small GTPase has been identified as a DNA damage-inducible gene. RhoB is involved in epidermal growth factor (EGF) receptor trafficking, cytoskeletal organization, cell transformation, and survival. We have analyzed the regulation of RhoB and elucidated its role in the cellular response of HaCaT keratinocytes to relevant environmental UVB irradiation. We report here that the activated GTP-bound form of RhoB is increased rapidly within 5 min of exposure to UVB, and then RhoB protein levels increased concomitantly with EGF receptor (EGFR) activation. Inhibition of UVB-induced EGFR activation prevents RhoB protein expression and AKT phosphorylation but not the early activation of RhoB. Blocking UVB-induced RhoB expression with specific small interfering RNAs inhibits AKT and glycogen synthase kinase-3␤ phosphorylation through inhibition of EGFR expression. Moreover, down-regulation of RhoB potentiates UVB-induced cell apoptosis. In contrast, RhoB overexpression protects keratinocytes against UVB-induced apoptosis. These results indicated that RhoB is regulated upon UVB exposure by a two-step process consisting of an early EGFR-independent RhoB activation followed by an EGFR-dependent induction of RhoB expression. Moreover, we have demonstrated that RhoB is essential in regulating keratinocyte cell survival after UVB exposure, suggesting its potential role in photocarcinogenesis.Among solar UV radiation that reaches the surface of the earth, UVB wavelengths are the most energetic. Directly absorbed by DNA and proteins, they account for much of the damaging biological effects of UV irradiation including premature skin aging and cancer (1). UVB acts as a carcinogen through both DNA damage and epigenetic effects. The initiation process in UVB-induced skin carcinogenesis involves UV light-induced DNA damage such as p53 or HA-ras mutations and represents an irreversible process (reviewed in Ref. 2). By contrast, UVBmediated skin tumor promotion involves the clonal expansion of initiated cells through activation of numerous signal transduction pathways, such as the MAPK 2 signaling cascades that coordinate cell cycle arrest, regulation of DNA repair, and apoptosis (3).It appears evident that after UVB exposure, the cell needs to integrate two opposing responses, with the final fate of keratinocytes depending on the balance between pro-and anti-apoptotic pathways. On the one hand, eliminating cells with excessive DNA damage is essential to preserve the health of the skin. UV-induced apoptosis plays this role through a complex mechanism triggered in three independent ways, via DNA damage-induced activation of caspas...

Effect of UVB irradiation on microRNA expression in mouse epidermis

“…These findings suggest that mmu-miR-223 is sensitive to UVB and elicits a direct or indirect effect of UVB-induced inflammation. RhoB, a predicted target of miR-223, has been reported to protect human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling (19), suggesting that miR-223 plays a role in regulating keratinocyte survival following UVB exposure. Little is known about the function of miR-188-5p and miR-22.…”

RhoB regulates uPAR signalling

“…RhoB activation is also rapidly stimulated by exposure to ultra violet (UVB) and ionizing radiation, which, similar to ATF, subsequently increase RhoB protein levels (Canguilhem et al, 2005;Monferran et al, 2008). The activation of RhoB by uPAR could be mediated by integrins as co-receptors for uPAR.…”

RhoB Promotes γH2AX Dephosphorylation and DNA Double-Strand Break Repair

“…Besides its well-established roles, RhoB emerged as an early DNA damage-inducible gene. RhoB is readily induced in response to various genotoxic agents, including UV and cisplatin (2,3), although the molecular mechanisms of induction and functional relevance remain unclear. RhoB also differs from other Rho proteins, as it possesses tumor suppressor functions.…”

“…U2OS cells were cotransfected with a plasmid encoding the RhoB promoter linked to the firefly luciferase reporter gene and a plasmid encoding the cytomegalovirus (CMV) promoter linked to the Renilla luciferase reporter gene (internal control), as described previously (2). Luciferase activities were measured 48 h after transfection by using the Dual Luciferase assay system (Promega), and results were expressed as the ratio of the activity of the firefly luciferase to the activity of the Renilla luciferase.…”

“…Amplification and deletion were considered significant events when at least 5 consecutive probes had a log 2 value of Ն0.3 (amplification) or a log 2 value of ՅϪ0.4 (deletion). We determined the genomic instability index with the formula (number of deletions ϩ number of amplifications) 2 /number of altered chromosomes, as previously described (32). Sexual chromosomes were excluded from the analysis.…”